Investigation of a novel supramolecular organisation of the oligomeric mucins MUC5AC MUC5B and MUC2

A layer of mucus is a common means of protecting the delicate exposed epithelial surfaces of an organism against its environment and a range of biological, chemical and physical insults. We have discovered that the gel-like characteristics of mucus in saliva and now potentially in other mucus gels (e.g. the respiratory, gastrointestinal and reproductive tracts) are caused by calcium binding in the large structural glycoproteins (mucins). Our evidence shows that this involves protein parts of the mucin linking mucin molecules together. Therefore we will identify the part(s) of the mucin responsible for calcium-binding by testing different parts of the molecule prepared separately and also in fragments of mucin purified from saliva. The details of how it functions and where it occurs in the structure of the mucin will be determined. We will also investigate whether mucins can be modified to remove or inactivate their calcium binding properties. This calcium dependent cross-link activity that we have detected in mucin is newly discovered and it is a potentially important mechanism contributing to the function of this vital protective barrier. Furthermore, an improved understanding of the organisation of this barrier has important implications for treatment of common diseases involving mucus overproduction (e.g. asthma and cystic fibrosis) and improved strategies for the delivery to and across epithelial surfaces of pharmaceuticals and gene therapy agents.

We have recently discovered a calcium dependent, reversible cross-link in MUC5B mucin from saliva, which increases the viscosity, greatly increases the average molecular weight and reduces the apparent porosity of the oligomeric mucin network. The activity was dependent on a single calcium binding site on each mucin monomer. This is a most important new finding as a mucus layer is a common means of protecting epithelial surfaces. Mucins are the major component responsible for the physical properties of the mucus gel, which are essential for its function. The properties have been interpreted until now as predominantly due to entanglement of the long, high molecular weight oligomeric mucins. We now have preliminary evidence that a comparable calcium dependent organisation is a feature of related oligomeric mucins namely MUC2 and MUC5AC. These two mucins are predominant gel-forming mucins in respiratory tract and gastrointestinal tract mucus. Using a range of biochemical/biophysical and molecular biology techniques this proposal will investigate the hypothesis that the gel-forming properties of oligomeric mucin is based on the presence of calcium binding protein domains that form reversible links between these oligomeric glycoproteins. Our research has identified the calcium-binding domain in a 120kDa N-terminal domain of MUC5B recombinantly expressed in mammalian cells and the position of the site(s) will be further investigated by expression of smaller mucin sub-domains. The number of binding sites per mucin monomer will be confirmed and the ligand specificity, binding affinity and stoichiometry of interaction will be determined. The calcium dependent property of these 3 oligomeric mucins will be investigated as a mechanism of heterologous interaction between mucin family members. We will also investigate if alternative gene splicing and proteolytic processing provide mechanisms for regulating their supramolecular organisation and hence mucus properties. This project will thus identify the molecular basis of this novel supramolecular organisation of mucus gels. This research will characterise and evaluate how this newly discovered mechanism of reversible supramolecular organisation controls their physical properties. The results of this study will identify possible strategies for controlling the excessive viscoelastic properties of mucus associated with various pathologies and methods to enhance the delivery of pharmaceuticals and gene therapy across epithelial surfaces.